title: MiR-133a induces apoptosis through direct regulation of GSTP1 in bladder cancer cell lines.
abstract: OBJECTIVE
  We previously demonstrated that miR-133a is a tumor-suppressive microRNA (miRNA) and is commonly down-regulated in human bladder cancer (BC). The aim of this study is to determine a novel oncogenic gene targeted by miR-133a in BC.
  
  
  METHODS
  To identify genes targeted by miR-133a, an oligo-microarray analysis was performed using the miR-133a-transfected BC cell lines. For gain/loss-of-function studies, miR-133a/si-glutathione S-transferase π1 (GSTP1)-transfectants were subjected to XTT assay and flow cytometry to evaluate their cell viability and apoptosis status. The luciferase reporter assay was used to confirm the actual binding sites between miR-133a and GSTP1 mRNA. The mRNA and protein expression of GSTP1 in BC cell lines and clinical samples were evaluated by real-time RT-PCR and Western blot, respectively.
  
  
  RESULTS
  MiR-133a transfection induced cell viability inhibition and apoptosis in BC cell lines. We focused on the GSTP1 gene that was the top 7 down-regulated one in the gene profile from the miR-133a-transfectants. MiR-133a transfection repressed expression levels of mRNA and protein levels of GSTP1. A luciferase reporter assay suggested that the actual binding may occur between miR-133a and GSTP1 mRNA. Cell viability inhibition and apoptosis were induced in the si-GSTP1 transfectants compared with the controls (P < 0.005). GSTP1 mRNA expression levels in 43 clinical BCs were significantly higher than those in eight normal bladder epitheliums (P = 0.0277).
  
  
  CONCLUSIONS
  Our data suggest that tumor suppressive miR-133a directly regulated oncogenic GSTP1 gene in BC, and that an anti-apoptotic effect mediated by GSTP1 is maintained by miR-133a down-regulation in human BC.
authors.affiliation: Department of Urology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.
authors.forename: Yousuke
authors.initials: Y
authors.lastname: Uchida
authors.forename: Takeshi
authors.initials: T
authors.lastname: Chiyomaru
authors.forename: Hideki
authors.initials: H
authors.lastname: Enokida
authors.forename: Kazumori
authors.initials: K
authors.lastname: Kawakami
authors.forename: Shuichi
authors.initials: S
authors.lastname: Tatarano
authors.forename: Kazuya
authors.initials: K
authors.lastname: Kawahara
authors.forename: Kenryu
authors.initials: K
authors.lastname: Nishiyama
authors.forename: Naohiko
authors.initials: N
authors.lastname: Seki
authors.forename: Masayuki
authors.initials: M
authors.lastname: Nakagawa
mesh_terms: D017209:Apoptosis; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D049109:Cell Proliferation; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D051549:Glutathione S-Transferase pi / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism / Q000556:secondary*; D008297:Male; D035683:MicroRNAs / Q000235:genetics*; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis; D011471:Prostatic Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D001749:Urinary Bladder Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*
chemical_list: D014408:Biomarkers, Tumor; C548234:MIRN133 microRNA, human; D035683:MicroRNAs; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D008156:Luciferases; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi